Photoperiod Genes Contribute to Daylength-Sensing and Breeding in Rice

Rice (Oryza sativa L.), one of the most important food crops worldwide, is a facultative short-day (SD) plant in which flowering is modulated by seasonal and temperature cues. The photoperiodic molecular network is the core network for regulating flowering in rice, and is composed of photoreceptors, a circadian clock, a photoperiodic flowering core module, and florigen genes. The Hd1-DTH8-Ghd7-PRR37 module, a photoperiodic flowering core module, improves the latitude adaptation through mediating the multiple daylength-sensing processes in rice. However, how the other photoperiod-related genes regulate daylength-sensing and latitude adaptation remains largely unknown. Here, we determined that mutations in the photoreceptor and circadian clock genes can generate different daylength-sensing processes. Furthermore, we measured the yield-related traits in various mutants, including the main panicle length, grains per panicle, seed-setting rate, hundred-grain weight, and yield per panicle. Our results showed that the prr37, elf3-1 and ehd1 mutants can change the daylength-sensing processes and exhibit longer main panicle lengths and more grains per panicle. Hence, the PRR37, ELF3-1 and Ehd1 locus has excellent potential for latitude adaptation and production improvement in rice breeding. In summary, this study systematically explored how vital elements of the photoperiod network regulate daylength sensing and yield traits, providing critical information for their breeding applications.


Introduction
The initiation of flowering, when plants transition from vegetative to reproductive growth, is one of the most crucial developmental decisions of the plant life cycle [1,2]. Flowering time is determined by a combination of endogenous genetic components and external environmental factors, such as photoperiod (daylength) and temperature [2]. Photoperiod induces flowering through three steps: the perception of the light signal, the regulation of the circadian clock, and flowering initiation [1,3].
Plants sense external light signals through different photoreceptors and transmit these light signals to the downstream circadian oscillator, which consists of a series of transcription−translation feedback loops, including the morning-phased proteins CCA1 (CIRCADIAN CLOCK ASSOCIATED 1), LHY (LATE ELONGATED HYPOCOTYL), RVE8 (REVEILLE 8), LNK1 (NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED GENE 1), LNK2, PRR9 (PSEUDO-RESPONSE REGULATOR 9), and PRR7, and the evening-phased genes TOC1 (TIMING OF CAB2 EXPRESSON 1), ELF3 (EARLY FLOWERING 3), ELF4, LUX (LUX ARHYTHMO), and GI (GIGANTEA) [4,5]. The evening complex (EC) is composed of ELF3, ELF4, and the DNA-binding protein LUX, which together form a transcriptional binding to the Hd1 and Ghd7 promoters to repress their transcription [29]. ELF3 is a flowering repressor in Arabidopsis, but OsELF3-1 promotes rice flowering by reducing the expression of Ghd7 [30]. A defect in OsLUX causes extremely late flowering and lower yields, while Oself4-2 mutants flower late under LD conditions, indicating that OsEC is required for the circadian clock to regulate the flowering time in rice [29,31]. The OsPRR gene family, encoding core components of the circadian clock, plays an important role in regulating photoperiodic flowering in rice [18,[32][33][34][35]. OsPRR37 delays flowering by negatively regulating Ehd1 and Hd3a expression under LD conditions [33,34]. Knocking out OsPRR73 led to early flowering under LD, but no change under SD conditions [35]. At the same time, the grain size and yield of the Osprr73 mutant were significantly reduced under salt stress conditions due to the lower salt tolerance displayed by this mutant [36].
Natural variation in core flowering regulatory genes is widely used in rice breeding, while photoreceptor genes and circadian clock-related genes have rarely been exploited. This study demonstrates that mutations in the photoreceptor and circadian clock genes generate different daylength-sensing processes. The se5 mutant, because it lacks active phytochromes, is deficient in photoperiodic responses and exhibits an early flowering phenotype and lower yield than the wild type. Mutation in ELF3-1 and Ehd1 can change the daylength-sensing processes and exhibit longer main panicle lengths and more grains per panicle. In addition, we identified single nucleotide polymorphisms (SNPs) or insertion/deletions (InDels) that introduced frameshifts, or large fragment deletions, in Hd1, DTH8, Ghd7, and PRR37. By contrast, we detected no frameshifts or InDel polymorphisms in SE5, OsGI, Ehd1, or ELF3-1 among 115 rice germplasms. Collectively, these findings provide critical information for breeding applications of photoperiod genes.

Photoperiod Genes Alter Daylength-Sensing in Rice
In our previous study, we developed a daylength-sensing-based environment adaptation simulator (DEAS) to forecast rice latitude adaptation via the transcriptional dynamics of florigen genes at different latitudes [16]. To assess whether loss-of-function alleles in photoperiod genes might affect daylength sensing, we measured the expression levels of Hd3a and RFT1 under various daylengths (daylength-sensing processes) in the rice cultivar Dongjin (DJ, wild type), Nipponbare (Nip, wild type), as well as the mutants Osgi, prr37, and elf3-1, grown under various daylengths (DEAS step 1). The DJ and Nip seedlings sensed a critical daylength (threshold = 13.5 h), as the expression of Hd3a and RFT1 was only induced when the daylength fell below 13.5 h (Figure 1a,b). In the prr37 mutant, the Hd3a expression was lower than in the DJ under daylengths shorter than 13.5 h, but higher for daylengths longer than 13.5 h (Figure 1a). RFT1 expression was also lower than that of DJ at photoperiods shorter than 13 h in prr37 mutant, but was higher at 13.5 h and 14 h daylengths, and became undetectable in the DJ and the prr37 mutant under a 15 h daylength ( Figure 1b). The prr37 mutant exhibited gradual daylength sensing for Hd3a expression and critical daylength sensing (threshold = 15 h) for RFT1 expression. Compared to DJ, the Hd3a and RFT1 expression was lower in elf3-1 when daylength was shorter than 13.5 h, but comparably lower in DJ and elf3-1 when the daylength was longer than 14 h. The elf3-1 mutant sensed a fine-tuned critical daylength threshold (14 h). However, the expression of RFT1 in the Osgi mutant was higher than that of elf3-1 under a 14 h daylength, and the expression of Hd3a and RFT1 was broadly similar in the Osgi and elf3-1 seedlings at the other daylengths tested (Figure 1a,b). The Osgi mutant exhibited critical daylength sensing for Hd3a and RFT1 expression, and the threshold was 14 and 15 h, respectively. These results indicate that mutations in circadian clock genes lead to changes in the daylength-sensing, to varying degrees, in rice. Ehd1 is a hub component in rice photoperiodic flowering and promotes flowering both LD and SD conditions [18,37]. We determined that the ehd1 and elf3-1 mutants exh the same daylength-sensing processes in DEAS (Figure 1a,b). The first step of photope odic flowering is the perception of a light signal. SE5 encodes a heme oxygenase that p ticipates in the chromophore biosynthesis for the red/far-red light photoreceptors phy chromes (phys) [38]. In the se5 mutant, Hd3a and RFT1 were expressed at high levels gardless of the photoperiod (Figure 1a,b), as was previously reported for the phyAph and phyAphyC double mutants [39]. Taken together, these results suggest that mutatio in the circadian clock genes and photoreceptor genes greatly affect the expression of orthologs (Hd3a or RFT1) when rice plants are grown under different daylengths.

The DEAS Couples Latitude Adaptation and Daylength Sensing
The latitude adaptation means that crops adapt to a given latitude when they c complete their entire growth period in a specific ecological environment. To explore relationship between latitude adaptation and daylength sensing, we planted DJ, prr Osgi, elf3-1, ehd1, Nip, and se5 in regions with latitudes of 30°41′N and 24°36′N. We a planted DJ, elf3-1, ehd1, Nip, and se5 at 41°40′N. We measured the daylengths betwe Ehd1 is a hub component in rice photoperiodic flowering and promotes flowering in both LD and SD conditions [18,37]. We determined that the ehd1 and elf3-1 mutants exhibit the same daylength-sensing processes in DEAS (Figure 1a,b). The first step of photoperiodic flowering is the perception of a light signal. SE5 encodes a heme oxygenase that participates in the chromophore biosynthesis for the red/far-red light photoreceptors phytochromes (phys) [38]. In the se5 mutant, Hd3a and RFT1 were expressed at high levels regardless of the photoperiod (Figure 1a,b), as was previously reported for the phyAphyB and phyAphyC double mutants [39]. Taken together, these results suggest that mutations in the circadian clock genes and photoreceptor genes greatly affect the expression of FT orthologs (Hd3a or RFT1) when rice plants are grown under different daylengths.

The DEAS Couples Latitude Adaptation and Daylength Sensing
The latitude adaptation means that crops adapt to a given latitude when they can complete their entire growth period in a specific ecological environment. To explore the relationship between latitude adaptation and daylength sensing, we planted DJ, prr37, Osgi, elf3-1, ehd1, Nip, and se5 in regions with latitudes of 30 • 41 N and 24 • 36 N. We also planted DJ, elf3-1, ehd1, Nip, and se5 at 41 • 40 N. We measured the daylengths between March 1st and October 11th at three latitudes (Figures 2, S2 and S3). We previously established that, based on the expression of Hd3a (or RFT1) under various daylengths in DEAS step1, DEAS Plants 2023, 12, 899 5 of 14 step 2 can use the daylength dynamics at a given latitude to infer an expression heatmap for Hd3a (or RFT1) at that latitude [16]. We thus produced expression heatmaps for Hd3a and RFT1 in DJ, prr37, Osgi, elf3-1, ehd1, Nip, and se5 at the two locations with latitudes of 30 • 41 N and 24 • 36 N (Figures 2 and S2) using the measured expression levels of Hd3a and RFT1 in DEAS step 1 (Figure 1a,b).
ts 2023, 12, x FOR PEER REVIEW 5 of expression heatmap for Hd3a (or RFT1) at that latitude [16]. We thus produced express heatmaps for Hd3a and RFT1 in DJ, prr37, Osgi, elf3-1, ehd1, Nip, and se5 at the two lo tions with latitudes of 30°41′N and 24°36′N ( Figure 2 and Figure S2) using the measur expression levels of Hd3a and RFT1 in DEAS step 1 (Figure 1a,b). . We speculate that the daylength at lower latitudes is shorter than at higher latitudes, and that the temperatures differ among the three regions. These results suggest that rice flowering time is related to number of days of transcriptional inactivation, low transcriptional activation, and hi transcriptional activation of Hd3a and RFT1.  (Figure 2a-d). At the same time, we predicted a high transcriptional activation of Hd3a and RFT1 throughout the growth period in se5, but the flowering times were different among the three regions (Figures 2k,l and S3d). We speculate that the daylength at the lower latitudes is shorter than at higher latitudes, and that the temperatures differed among the three regions. These results suggest that rice flowering time is related to the number of

Photoperiod Genes Affect Rice Yield
Agronomic traits, such as the number of grains per panicle, hundred-grain weight, and seed-setting rate, are closely related to rice yield [40]. In addition to flowering time, we wonder if photoperiod genes affect rice yield. To this end, we measured the main panicle length, number of grains per panicle, seed-setting rate, hundred-grain weight, grain length, grain width, and yield per panicle of DJ, prr37, Osgi, elf3-1, ehd1, Nip, and se5 plants (Figures 3 and S4). We observed that prr37, elf3-1, and ehd1 mutants have longer growth periods, accompanied by longer main panicles and more grains per panicle (Figures 2d,f,h and 3a,e,f). By contrast, the se5 mutant had the shorter growth period, shorter main panicles, fewer grains per panicle, and lighter yield per panicle (Figures 2l, 3a,e,f and S4). Notably, mutations in the core circadian clock genes ELF3-1 and OsGI led to lower setting rates (Figure 3g), particularly OsGI, as the Osgi mutant showed low values for the hundred-grain weight and yield per panicle (Figures 3h and S4). In previous reports, the Osgi mutant was shown to display reduced fertility under atypical growing conditions with late transplanting dates [41]. Mutations in PRR37, OsGI, ELF3-1, Ehd1, and SE5, caused almost no changes in the grain length or grain width (Figure 3i,j). Importantly, the Osgi and se5 mutants had a lower yield per panicle, and the prr37 and ehd1 mutants had higher yields per panicle (Figures 3d and S4). Collectively, these results suggest that photoperiod genes not only change the daylength-sensing processes, but also the agronomic traits in rice.
ts 2023, 12, x FOR PEER REVIEW 6 of panicle length, number of grains per panicle, seed-setting rate, hundred-grain weig grain length, grain width, and yield per panicle of DJ, prr37, Osgi, elf3-1, ehd1, Nip, and plants (Figures 3 and S4). We observed that prr37, elf3-1, and ehd1 mutants have long growth periods, accompanied by longer main panicles and more grains per panicle (F ures 2d,f,h and 3a,e,f). By contrast, the se5 mutant had the shorter growth period, shor main panicles, fewer grains per panicle, and lighter yield per panicle (Figures 2l, 3a  and S4). Notably, mutations in the core circadian clock genes ELF3-1 and OsGI led to low setting rates (Figure 3g), particularly OsGI, as the Osgi mutant showed low values for t hundred-grain weight and yield per panicle (Figures 3h and S4). In previous reports, t Osgi mutant was shown to display reduced fertility under atypical growing conditio with late transplanting dates [41]. Mutations in PRR37, OsGI, ELF3-1, Ehd1, and S caused almost no changes in the grain length or grain width (Figure 3i,j). Importantly, t Osgi and se5 mutants had a lower yield per panicle, and the prr37 and ehd1 mutants h higher yields per panicle (Figures 3d and S4). Collectively, these results suggest that ph toperiod genes not only change the daylength-sensing processes, but also the agronom traits in rice.

Nucleotide Polymorphism of Photoperiod Genes in Rice cultivars
The Hd1-DTH8-Ghd7-PRR37 module regulates rice daylength-sensing in rice, a natural variation in the underlying photoperiod genes changes the daylength-sensi processes, which play a vital part in the adaptation of rice cultivars to multi-latitude , hundred-grain weight (h), grain length (i), and grain width (j) of DJ, prr37, elf3-1, Osgi, ehd1, Nip, and se5. Data are presented as means ± SD, n = 15 (e-g), or four biological replicates (h-j). Scale bars: 2 cm for main panicle; 5 mm for grain length; 5 mm for grain width; 1 cm for yield per panicle. The letters above each column indicate significant differences by Duncan's multiple range test (p < 0.05). All agronomic traits of the wild-type and mutant were collected under natural conditions in Xiamen.

Nucleotide Polymorphism of Photoperiod Genes in Rice cultivars
The Hd1-DTH8-Ghd7-PRR37 module regulates rice daylength-sensing in rice, and natural variation in the underlying photoperiod genes changes the daylength-sensing processes, which play a vital part in the adaptation of rice cultivars to multi-latitude regions [16,24]. To expand this analysis to photoperiod genes, we collected the DNA sequences for Hd1, DTH8, Ghd7, PRR37, SE5, OsGI, Ehd1, and ELF3-1 from 115 rice germplasm resources (70 indica, 30 japonica, 12 Aus, and 3 Bus) [42][43][44][45][46][47][48][49][50][51], and looked for polymorphisms ( Figure 4). Among the 115 rice materials, we identified 11 haplotypes for Hd1, of which seven (in 55 rice varieties) were functional, while the remaining four haplotypes (present in 60 rice varieties) were predicted to encode a non-functional protein due to the presence of frameshifts (Figure 4a). Likewise, we detected 12 haplotypes for DTH8, with 40 rice varieties harboring non-functional alleles, including 37 indica (Figure 4b). This result was consistent with our previous study, in which we determined that Hd1hd1 dth8dth8 is one of the major genotypes of indica hybrid rice in East Asia [16]. We also observed two nonfunctional haplotypes at Ghd7, one with a large fragment deletion and the other with early translation termination, accounting for 14.8% (or 17) of all accessions among the 115 germplasms (Figure 4c). We identified three non-functional variants in PRR37, two with frameshift mutations and the one with three amino acid substitutions (Figure 4d). Three of them (N214S, L462P, and P710L) were located at the conserved positions among their homologs, and a previous study suggested that these substitutions may affect the PRR37 function [34]. REVIEW 7 4b). This result was consistent with our previous study, in which we determined Hd1hd1 dth8dth8 is one of the major genotypes of indica hybrid rice in East Asia [16 also observed two non-functional haplotypes at Ghd7, one with a large fragment del and the other with early translation termination, accounting for 14.8% (or 17) of all a sions among the 115 germplasms (Figure 4c). We identified three non-functional var in PRR37, two with frameshift mutations and the one with three amino acid substitu (Figure 4d). Three of them (N214S, L462P, and P710L) were located at the conserve sitions among their homologs, and a previous study suggested that these substitu may affect the PRR37 function [34]. Notably, we detected no frameshift mutations or deletions in the coding seque of OsGI, ELF3-1, Ehd1, or SE5 among the 115 rice germplasms (Figure 4e-h). SE5 is h Notably, we detected no frameshift mutations or deletions in the coding sequences of OsGI, ELF3-1, Ehd1, or SE5 among the 115 rice germplasms (Figure 4e-h). SE5 is highly conserved, without any amino acid variation in the 115 germplasms (Figure 4h). One amino acid substitution (G219R) in the Golden2, Arabidopsis RESPONSE REGULATOR (ARR), and Chlamydomonas regulatory protein of P-starvation acclimatization response (Psr1) (GARP) domain of Ehd1 was previously demonstrated to lower the DNA-binding activity of Ehd1 [37,52]. However, this Ehd1 G219R allele is rare and was not represented in our panel of 115 varieties (Figure 4g). ELF3-1 isoforms can be divided into ELF3-1(L) and ELF3-1(S) (weak function) based on the amino acid at position 558. Overall, 79.1% (or 91) of the 115 varieties produce ELF3-1(S) (Figure 4f). Compared to ELF3-1(L), ELF3-1(S) delayed rice flowering under LD conditions [53]. The japonica varieties carrying ELF3-1(L) occur at higher latitudes, while the varieties carrying ELF3-1(S) are found at lower latitudes [54].

Discussion
The timing of flowering is a key agronomic trait that determines the latitudinal adaptability and planting seasons of rice cultivars. A suitable flowering time allows rice plants to make full use of light and temperature resources to maximize yield [16,55]. Indica rice cultivars with the genotype Hd1 DTH8 Ghd7 PRR37 are characterized by extremely low expression of florigen when the daylength is longer than 13 h, and fail to flower under natural LD conditions [24]. However, our results also indicate that the time to flowering of DJ and Nip (japonica, Hd1 DTH8 Ghd7 PRR37 genotype) was about 145 days at 41 • 40 N ( Figure S3a), which exceeds the suitable growing season. The selection of rice varieties adapted to various latitudes has capitalized on the variations in the core flowering regulatory genes Hd1, Ghd7, DTH8, and PRR37 [16,17,21,34]. A loss-of-function mutation of any of these four genes results in reduced PS, and different allelic combinations at these four genes exhibit diverse degrees of PS, ranging between strong PS and complete photoperiod insensitivity [16,17,21,55]. The wild rice O. rufipogon harbors functional alleles at Hd1, DTH8, Ghd7, and PRR37 (HDGP) and thus possesses strong PS [55]. In rice, the natural variation of core photoperiod genes is an important molecular basis for latitudinal expansion. In this study, we found frameshift mutations or deletions in the coding sequences of Hd1, DTH8, Ghd7, and PRR37 among some rice varieties (Figure 4). In addition, in the Heilongjiang Province of China (situated at relatively high latitudes), many local modern japonica varieties carry non-functional hd1/ghd7/prr37 (hgp) and weak functional DTH8 (or weak-functional DTH8/Ghd7 and non-functional hd1/prr37) alleles, resulting in weak PS to match the lower temperature and longer daylength of the region [55]. Very few hybrid rice varieties carry strong functional alleles at Hd1/DTH8/Ghd7, because their combination produces strong PS and extremely late flowering time [55]. In addition to the four core flowering regulatory genes (Figure 4a-d), several minor genes are also used in breeding, such as Hd16, DTH2, OsMADS56, and RFT1 [56][57][58][59].
Plants perceive light signals through various photoreceptors, such as the red/far-red light receptor phytochromes and blue light receptor cryptochromes [1]. There are three phytochromes in rice: OsPHYA, OsPHYB and OsPHYC. Under natural LD conditions, phyB and phyC single mutants show an early-flowering phenotype, while phyA does not change the flowering time [60]. The phyAphyC and phyAphyB double mutants flower very early under natural LDs and exhibit daylength insensitivity [39,60], similar to the daylengthsensing processes of the se5 mutant (Figure 1a,b). Meanwhile, the seed setting rate of the phyAphyB double mutant is significantly decreased [61]. There are three cryptochromes in rice, OsCRY1a, OsCRY1b, and OsCRY2, but only OsCRY2 promotes flowering under both SD and LD conditions [62]. In addition, we revealed that the yield per panicle of the Osgi and se5 mutants was significantly decreased compared to that in the wild type (Figures 3d and S4). These findings indicate that photoreceptor genes and circadian clock genes can not only regulate daylength-sensing processes (Figure 1), thereby changing the flowering time and latitude adaptation, but also affect yields in rice (Figures 3 and S4).
Soybean is a typical SD plant, and the suitable flowering time guarantees high yields. E1, a flowering repressor in soybean, is a hub of the photoperiodic flowering network, which represses the expression of the florigen genes, GmFT2a and GmFT5a, to delay flowering. E2 (GmGI), E3 (GmphyA3) and E4 (GmphyA2) repress flowering and induce the expression of E1. The natural variation of E1, E3 and E4 change the soybean daylength-sensing processes, expanding the planting area to higher latitudes [63][64][65]. The soybean J gene, as the ortholog of Arabidopsis ELF3, represses the expression of E1 to promote soybean flowering. The J gene of soybean and the ELF3-1 gene of rice function similarly, both as flowering activators (Figure 2a,b,e,f). Mutation of the soybean J locus prolongs the vegetative growth period and improves its adaptation to the tropics [66]. In the process of soybean domestication, the mutation in Tof11 (PRR3b) and Tof12 (PRR3a) contributed to flowering, and the plants matured earlier, improving the higher latitude adaptation [67]. Compared to wild soybean, the cultivars in Northeast China carry higher abundant non-functional tof12 [67]. Tof5 is also related to soybean higher latitude adaptation, which promotes flowering via inducing the expression of FT2a and FT5a [68]. The novel locus Tof16 (a homolog of LHY) is a repressor of E1 and enabled the soybean to migrate from its temperate origin to the tropics; more than 80% of accessions in low-latitude areas contain loss-of function tof16 and j [69]. Haplotypes of GmFT2a and GmFT5a are also involved in the soybean flowering phenotypes, maturity time and geographical distributions [65,70].
Maize was domesticated from teosinte (Zea mays ssp. parviglumis) and originated in southwestern Mexico [71]. ZCN8 is homologous to Arabidopsis FT, the SNP-1245 and InDel-2339 in the ZCN8 promoter regions play an important role in maize expanding to high latitudes [72]. ZmCCT9 and ZmCCT10 contain a CCT domain, homologous to rice Ghd7, repress the expression of ZCN8, and delay flowering under LD conditions [73,74]. The transposon insertion upstream of ZmCCT9 and ZmCCT10 reduces their expression and accelerates the process of maize adapting to higher latitudes [73,74].
There are various photoperiod genes involved in the latitudinal adaptation of rice, soybean, and maize. In rice and maize, transcription factors are widely selected in breeding, such as Hd1, DTH8, Ghd7 (Figure 4), ZmCCT9, and ZmCCT10. However, photoreceptors (E3 and E4) and circadian clock genes (Tof11, Tof12, and J) are widely used in soybean breeding. In lower latitudes, local farmers prefer double or triple cropping rice throughout the year to harvest more grain. Mutations in Ehd1 or ELF3-1 prolong the growth period in rice, so they are probably not suitable for rice multiple cropping. Soybean from temperate regions introduced to lower latitudes flower early and have an extremely low yield. Natural variation at the soybean J (ELF3) locus extends the vegetative phase under inductive SD conditions and increases yield [66].
Diverse combinations of the Hd1, DTH8, Ghd7, and PRR37 genes mediate the multiple daylength-sensing processes to improve the latitude adaptation [16]. However, optimal cropping modes coupled with proper daylength-sensing processes can enhance rice multilatitude adaptation [24]. Mutations in some circadian clock genes and photoreceptor genes greatly affect the daylength-sensing processes and yield-related traits, resulting in a mismatch between the growth period of crops and the arable season. Notably, our results indicated that the ehd1 and elf3-1 mutants had longer main panicle lengths and more grains per panicle than DJ ( Figure S4). Based on our findings and previous research [75,76], mutations in Ehd1 and ELF3-1 improved the grain and yield (Figures 3 and S4). Hence, with global warming, as the growing season length will be extended, perhaps the Ehd1 and ELF3-1 locus could be applied to rice breeding in the future.
The rice (Oryza sativa) subspecies japonica DJ and Nip were used as the wild type. The T-DNA insertion mutants elf3-1 and Osgi were previously reported [30,78]. The se5 mutant with a nucleotide mutation caused an early termination. All of the recombinant vectors and mutants were confirmed by PCR or sequencing. The DNA sequencing results are shown in Supplementary Figure S1. The primers are listed in Table S1.

Plant Growth Conditions
For the RT-qPCR assay, rice seedlings were grown in growth chambers with 28 • C and a relative humidity of~70% for 35 days. Six growth chambers were used with 12, 13, 13.5, 14 and 15 h daylengths, respectively. The light in the growth chambers was supplied by light-emitting diodes (Sanan Sino-Science, Xiamen, China). For the flowering phenotypic assays in the field, seeds were sown in Xiamen (24 •

Analysis of Gene Expression
On day 35, all of the rice seedling leaves were harvested 3 h after dawn. The total RNA was isolated from the samples with an Eastep Super Total RNA Extraction Kit (Promega, Beijing, China) and were reverse transcribed with the GoScript Reverse Transcription Mix using oligo(dT) (Promega, Beijing, China), according to the manufacturer's instructions. Real-time quantitative PCR (RT-qPCR) was performed using the SYBR Green PCR (Bio-Rad) method on a CFX Connect TM Real-Time PCR System (Bio-Rad, California, USA), following the manufacturer's instructions. The transcriptional data for Hd3a and RFT1 were then collected. When the expression of Hd3a and RFT1 differed by more than 10-fold between adjacent daylengths, we categorized this expression pattern as critical daylength sensing. When the florigen gene expression changed gradually with the change in the daylength and the difference between the adjacent daylengths was less than 10-fold, we categorized this expression pattern as representing gradual daylength sensing. The primers used for the RT-qPCR are listed in Table S1.

Florigen Gene Expression Heatmap
First, we divided the expression of florigen genes into three levels in the inferred Hd3a and RFT1 expression profiles. When the Hd3a or RFT1 expression was <10 × 10 −4 , we defined them as being inactivated. When the 10 × 10 −4 <Hd3a or RFT1 expression <100 × 10 −4 , we defined them as being weakly inactivated. When the 100 × 10 −4 <Hd3a or RFT1 expression, we defined them as being highly inactivated. Subsequently, we obtained the florigen gene-expression heatmap according to the methods previously described [16,24]. The daylength data for different latitudes were collected using the Rise and Set Times app developed by S. Vdovenko (http://www.lifewaresolutions.com/ (accessed on 30 January 2020)).

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants12040899/s1, Figure S1: Sequence of different mutant alleles., Figure S2: The effect of latitude and genotype on dynamics of RFT1 transcript levels., Figure S3: The effect of latitude and genotype on dynamics of Hd3a transcript levels., Figure S4: Yield per panicle of wild-type and mutant; Table S1: The primers used in this study.